GlmU

ABSTRACT

The invention provides GlmU polypeptides and polynucleotides encoding GlmU polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing GlmU polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims the benefit of Provisional Application Ser. No.60/050,996, filed, Jun. 26, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to novel polynucleotides and polypeptides of the GlmU family,hereinafter referred to as "GlmU".

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This phenomenon has createda demand for both new anti-microbial agents, vaccines, and diagnostictests for this organism.

N-Acetylglucosamine-1-Phosphate Uridyltransferase (GlmU) catalyses theformation of UDP-N-acetylglucosamine, an essential precursor for cellwall peptidoglycan in all bacteria and of lipopolysaccharide andenterobacterial common antigen in gram negatives. The enzyme has beenpurified from Escherichia coli and is bifunctional, also catalyzing thepreceeding step of N-acetylation of glucosamine-1-phosphate(Mengin-Lecreulx, D. and van Heijenoort, J, J.Bacteriol. 176: 5788-5795[1994]). It is possible to block the acetyltransferase activity but notthe uridyl transferase activity with thiol inhibitors, suggesting thatthe enzyme may have two domains. The gene, glmU, encoding the enzyme hasbeen cloned from E. coli (Mengin-Lecreulx, D. and van Heijenoort, J,J.Bacteriol. 175: 6150-6157 [1993]) and its counterpart in Bacillussubtilis (gcaD) has also been identified (Hove-Jensen B, J.Bacteriol.174: 6852-6 [1992]).

The essential nature of the gene product of gcaD is demonstrated bytemperature sensitive mutants of Bacillus subtilis which are unable tomake active enzyme and stop growing at the restrictive temperature(Hove-Jensen [1992]). Inhibitors of these proteins therefore haveutility in anti-bacterial therapy. The discovery of the gene from thehuman pathogen Streptococcus pneumoniae corresponding to gcaD/GlmUpermits production of the enzyme which can be used to screen for novelantibiotics.

Clearly, there exists a need for factors, such as the GlmU embodimentsof the invention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists to find ways to prevent,ameliorate or correct such infection, dysfunction and disease.

Certain of the polypeptides of the invention possess amino acid sequencehomology to a known GlmU protein from Bacillus subtilis. See PIRdatabase S66050; Genembl D26185; and Swissprot P14192. Also see NILSSOND., HOVE-JENSEN B., ARNVIG K. MOL. GEN. GENET. 218:565-571 (1989);OGASAWARA N., NAKAI S., YOSHIKAWA H. DNA RES. 1:1-14 (1994).

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel GlmU polypeptides by homology between the amino acidsequence set out in Table 1 [SEQ ID NO: 2 or 4] and a known amino acidsequence or sequences of other proteins such as GlmU from Bacillussubtilis protein.

It is a further object of the invention to provide polynucleotides thatencode GlmU polypeptides, particularly polynucleotides that encode thepolypeptide herein designated GlmU.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding GlmU polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO:1 or 3] which includes a fulllength gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel GlmU protein from Streptococcus pneumoniae comprising the aminoacid sequence of Table 1 [SEQ ID NO:2 or 4], or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Streptococcus pneumoniae 0100993 strain contained in thedeposited strain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding GlmU, particularly Streptococcus pneumoniaeGlmU, including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of GlmU and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStreptococcus pneumoniae referred to herein as GlmU as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of GlmU polypeptide encoded by naturally occurring alleles ofthe GlmU gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned GlmU polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing GlmUexpression, treating disease, assaying genetic variation, andadministering a GlmU polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStreptococcus pneumoniae bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to GlmU polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against GlmU polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided GlmU agonists and antagonists, preferably bacteriostatic orbactericidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a GlmU polynucleotide or a GlmU polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to novel GlmU polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel GlmU of Streptococcuspneumoniae, which is related by amino acid sequence homology to GlmUpolypeptide from Bacillus subtilis. See PIR database S66050; GenemblD26185; and Swissprot P14192. Also see NILSSON D., HOVE-JENSEN B.,ARNVIG K. MOL. GEN. GENET. 218:565-571 (1989); OGASAWARA N., NAKAI S.,YOSHIKAWA H. DNA RES. 1:1-14 (1994). The invention relates especially toGlmU having the nucleotide and amino acid sequences set out in Table 1as SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and to the GlmUnucleotide sequences of the DNA in the deposited strain and amino acidsequences encoded thereby.

                                      TABLE 1                                     __________________________________________________________________________    GlmU Polynucleotide and Polypeptide Sequences                                 __________________________________________________________________________    (A) Sequences from Streptococcus pneumoniae                                     GlmU polynucleotide sequence [SEQ ID NO:1].                                 5'-1                                                                              AAAAGCCTGT GCTTCAANTC TTGTGCTATA TTGGATTTTT GTTTTAAACG                      51 ATTGGCTGTC ATTAAGTGGG CGATTAATGA TTAAAATGNA CATCATAATC                     101 CCAAAAAAAC TAAATAAAAT AAGTGGATGA ATTTGTTTTC TCATATCTTA                    151 TAATTCTACC CTAAAAATCA AAAAAAATCA AAAAAATGGG TTAAGGAAGA                    201 GACTTTAGAG CATTTTTTCA TTCAAGAGTG CGGAATGATT TGAAATATGG                    251 TATAATAAAA GGGAATTTCT ACAGAAAAGA GAAGATTATG TCAAATTTTG                    301 CCATTATTTT AGCAGCGGGT AAAGGGACTC GCATGAAATC TGATTTGCCA                    351 AAAGTTTTGC ACAAGGTTGC GGGTATTTCT ATGTTGGAAC ATGTTTTCCG                    401 TAGTGTGGGA GCTATCCAAC CTGAAAAGAC AGTAACAGTT GTAGGACACA                    451 AGGCAGAATT GGTTGAGGAG GTCTTGGCTG GACAGACAGA ATTTGTGACT                    501 CAATCTGAAC AGTTGGGAAC TGGTCATGCA GTTATGATGA CAGAGCCTAT                    551 CTTAGAAGGT TTGTCAGGAC ACACCTTGGT CATTGCAGGA GATACTCCTT                    601 TAATCACTGG TGAAAGCTTG AAAAACTTGA TTGATTTCCA TATCAATCAT                    651 AAAAATGTGG CCACTATCTT GACTGCTGAA ACGGATAATC CTTTTGGCTA                    701 TGGACGAATT GTTCGTAATG ACAATGCTGA GGTTCTTCGT ATGGTTGAGC                    751 AGAAGGATGC TACAGATTTT GAAAAGCAAA TCAAGGAAAT CAACACTGGA                    801 ACATACGTCT TTGACAACGA GCGTTTGTTT GAGGCTTTGA AAAATATCAA                    851 TACCAATAAC GCTCAAGGCG AATACTATAT TACAGACGTC ATTGGTATTT                    901 TCCGTGAAAC TGGTGAAAAA GTTGGCGCTT ATACTTTGAA AGATTTTGAT                    951 GAAAGTCTTG GGGTAAATGA CCGTGTGGCG CTTGCGACAG CTGAGTCAGT                    1001 TATGCGTCGT CGCATCAATC ATAAACACAT GGTCAACGGT GTTAGCTTTG                   1051 TCAATCCAAA AGCAACTTAT ATCGATATTG ATGTTGAGAT TGCTTCGGAA                   1101 GTTCAAATCG AAGCCAATGT TACCTTGAAA GGGCAAACGA AAATTGGTGC                   1151 TGAGACTGTT TTGACAAACG GTACTTATGT AGTGGACAGC ACTATCGGAG                   1201 CAGGAGCGGT CATTACCAAT TCTATGATTG AGGAAAGTAG TGTTGCAGAC                   1251 GGTGTGACAG TCGGTCCTTA TGCTCACATT CGTCCAAATT CAAGTCTGGG                   1301 TGCCCAAGTT CATATTGGTA ACTTTGTTGA GGTGAAAGGA TCTTCAATCG                   1351 GTGAGAATAC CAAGGCTGGT CATTTGACTT ATATCGGAAG CTGTGAAGTG                   1401 GGAAGCAACG TTAATTTCGG TGCTGGAACT ATTACAGTCA ACTATGACGG                   1451 CAAAAACAAA TACAAGACAG TCATTGGAGA CAATGTCTTT GTTGGTTCAA                   1501 ATTCAACCAT TATTGCACCA GTAGAACTTG GTGACAATTC CCTCGTTGGT                   1551 GCTGGTTCAA CTATTACTAA AGACGTGCCA GCAGATGCTA TTGCTATTGG                   1601 TCGCGGTCGT CAGATCAATA AAGACGAATA TGCAACACGT CTTCCTCATC                   1651 ATCCTAAGAA CCAGTAGGAG CCTATCATGG AGTTTGAAGA AAAAACGCTT                   1701 AGCCGAAAAG AAATCTATCA AGGACCAATA TTTAAACTGG TCCAAGATCA                   1751 GGTTGAATTA CCAGAAGGCA AGGGAACTGC CCAACGGGAT TTGATTTTCC                   1801 ACAATGGGGC TGTCTGTGTT TTAGCAGTAA CGGATGAACA AAAACTTATC                   1851 TTGGTCAAGC AGTACCGCAA AGCTATCGAG GCTGTCTTTT ACGAAATTTC                   1901 AGCCGGAAAA TTGGAAGTAG GAGAAAACAC AGCCCCTGTG GCAGCTGCCC                   1951 TTCGTGAATT AGAGGAAGAA ACAGCCTATA CAGGGAAATT AGAACTCTTG                   2001 TACGATTTTT ATTCAG-3'                                                   (B) Streptococcus pneumoniae GlmU polypeptide sequence                          deduced from the polynucleotide sequence                                      in this table [SEQ ID NO:2].                                                NH.sub.2 -1                                                                       MSNFAIILAA GKGTRMKSDL PKVLHKVAGI SMLEHVFRSV GAIQPEKTVT                      51 VVGHKAELVE EVLAGQTEFV TQSEQLGTGH AVMMTEPILE GLSGHTLVIA                     101 GDTPLITGES LKNLIDFHIN HKNVATILTA ETDNPFGYGR IVRNDNAEVL                    151 RMVEQKDATD FEKQIKEINT GTYVFDNERL FEALKNINTN NAQGEYYITD                    201 VIGIGRETGE KVGAYTLKDF DESLGVNDRV ALATAESVMR RRINHKHMVN                    251 GVSFVNPKAT YIDIDBEIAS EVQIEANVTL KGQTKIGAET VLTNGTYVVD                    301 STIGAGAVIT NSMIEESSVA DGVTVGPYAH IRPNSSLGAQ VHIGNFVEVK                    351 GSSIGENTKA GHLTYIGSCE VGSNVNFGAG TITVNYDHKN KYKTVIGDNV                    401 FVGSNSTIIA PVELGDNSLV GAGSTITKDV PADAIAIGRG RQINKDEYAT                    451 RLPHHPKNQ-COOH                                                          (C) Polynucleotide sequences comprising                                         Streptococcus pneumoniae GlmU ORF                                             sequence [SEQ ID NO:3].                                                     5'-1                                                                              TCATGCAGTT ATGATGACAG AGCCTATCTT AGAAGGTTTG TCAGGACACA                      51 CCTTGGTCAT TGCAGGAGAT ACTCCTTTAA TCACTGGTGA AAGCTTGAAA                     101 AACTTGATTG ATTTCCATAT CAATCATAAA AATGTGGCCA CTATCTTGAC                    151 TGCTGAAACG GATAATCCTT TTGGCTATGG ACGAATTGTT CGTAATGACA                    201 ATGCTGAGGT TCTTCGTATG GTTGAGCAGA AGGATGCTAC AGATTTTGAA                    251 AAGCAAATCA AGGAAATCAA CACTGGAACA TACGTCTTTG ACAACGAGCG                    301 TTTGTTTGAG GCTTTGAAAA ATATCAATAC CAATAACGCT CAAGGCGAAT                    351 ACTATATTAC AGACGTCATT GGTATTTTCC GTGAAACTGG TGAAAAAGTT                    401 GGCGCTTATA CTTTGAAAGA TTTTGATGAA AGTCTTGGGG TAAATGACCG                    451 TGTGGCGCTT GCGACAGCTG AGTCAGTTAT GCGTCGTCGC ATCAATCATA                    501 AACACATGGT CAACGGTGTT AGCTTTGTCA ATCCAAAAGC AACTTATATC                    551 GATATTGATG TTGAGATTGC TTCGGAAGTT CAAATCGAAG CCAATGTTAC                    601 CTTGAAAGGG CAAACGAAAA TTGGTGCTGA GACTGTTTTG ACAAACGGTA                    651 CTTATGTAGT GGACAGCACT ATCGGAGCAG GAGCGGTCAT TACCAATTCT                    701 ATGATTGAGG AAAGTAGTGT TGCAGACGGT GTGACAGTCG GTCCTTATGC                    751 TCACATTCGT CCAAATTCAA GTCTGGGTGC CCAAGTTCAT ATTGGTAACT                    801 TTGTTGAGGT GAAAGGATCT TCAATCGGTG AGAATACCAA GGCTGGTCAT                    851 TTGACTTATA TCGGAAGCTG TGAAGTGGGA AGCAACGTTA ATTTCGGTGC                    901 TGGAACTATT ACAGTCAACT ATGACGGCAA AAACAAATAC AAGACAGTCA                    951 TTGGAGACAA TGTCTTTGTT GGTTCAAATT CAACCATTAT TGCACCAGTA                    1001 GAACTTGGTG ACAATTCCCT CGTTGGTGCT GGTTCAACTA TTACTAAAGA                   1051 CGTGCCAGCA GATGCTATTG CTATTGGTCG CGGTCGTCAG ATCAATAAAG                   1101 ACGAATATGC AACACGTCTT CCTCATCATC CTAAGAACCA GTAGGAGCCT                   1151 ATCATGGAGT TTGAAGAAAA AACGCTTAGC CGAAAAGAAA TCTATCAAGG                   1201 ACCAATATTT AAACTGGTCC AAGATCAGGT TGAATTACCA GAAGGCAAGG                   1251 GAACTGCCCA ACGGGATTTG ATTTTCCACA ATGGGGCTGT CTGTGTTTTA-3'              (D) Streptococcus pneumoniae GlmU                                               polpeptide sequence deduced from the                                          polynucleotide ORF sequence in this tabel [SEQ ID NO:4].                    NH.sub.2 -1                                                                       HAVMMTEPIL EGLSGHTLVI AGDTPLITGE SLKNLIDFHI NHKNVATILT                      51 AETDNPFGYG RIVRNDNAEV LRMVEQKDAT DFEKQIKEIN TGTYVFDNER                     101 LFEALKNINT NNAQGEYYIT DVIGIFRETG EKVGAYTLKD FDESLGVNDR                    151 VALATAESVM RRRINHKHMV NGVSFVNPKA TYIDIDVEIA SEVQIEANVT                    201 LKGQTKIGAE TVLTNGTYVV DSTIGAGAVI TNSMIEESSV ADGVTVGPYA                    251 HIRPNSSLGA QVHIGNFVEV KGSSIGENTK AGHLTYIGSC EVGSNVNFGA                    301 GTITVNYDGK BKYKTVIGDN VFVGSNSTII APVELGDNSL VGAGSTITKD                    351 VPADAIAIGR GRQINKDEYA TRLPHHPKNQ-COOH                                   __________________________________________________________________________

Deposited Materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on 11 Apr. 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit. On 17 Apr.1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli wassimilarly deposited with the NCIMB and assigned deposit number 40800.The Streptococcus pneumoniae strain deposit is referred to herein as"the deposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length GlmU gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. § 112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2 or 4] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of GlmU, and also those which have at least 70% identity to apolypeptide of Table 1 [SEQ ID NO:1 or 3] or the relevant portion,preferably at least 80% identity to a polypeptide of Table 1 [SEQ IDNO:2 or 4and more preferably at least 90% similarity (more preferably atleast 90% identity) to a polypeptide of Table 1 [SEQ ID NO:2 or 4] andstill more preferably at least 95% similarity (still more preferably atleast 95% identity) to a polypeptide of Table 1 [SEQ ID NO:2 or 4] andalso include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

The invention also includes polypeptides of the formula:

    X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y

wherein, at the amino terminus, X is hydrogen, and at the carboxylterminus, Y is hydrogen or a metal, R₁ and R₃ are any amino acidresidue, m is an integer between 1 and 1000 or zero, n is an integerbetween 1 and 1000 or zero, and R₂ is an amino acid sequence of theinvention, particularly an amino acid sequence selected from Table 1. Inthe formula above R₂ is oriented so that its amino terminal residue isat the left, bound to R₁, and its carboxy terminal residue is at theright, bound to R₃. Any stretch of amino acid residues denoted by eitherR group, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with GlmU polypeptides fragments maybe "free-standing," or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2 or 4], or ofvariants thereof, such as a continuous series of residues that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus. Degradation forms of the polypeptides of theinvention in a host cell, particularly a Streptococcus pneumoniae, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of GlmU, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

In addition to the standard single and triple letter representations foramino acids, the term "X"or "Xaa" may also be used in describing certainpolypeptides of the invention. "X" and "Xaa" mean that any of the twentynaturally occuring amino acids may appear at such a designated positionin the polypeptide sequence.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the GlmU polypeptide havinga deduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4] andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1 or 3], a polynucleotide of the inventionencoding GlmU polypeptide may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using Streptococcus pneumoniae 0100993 cellsas starting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as asequence given in Table 1 [SEQ ID NO: 1 or 3], typically a library ofclones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E. colior some other suitable host is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using stringent conditions. By sequencing the individualclones thus identified with sequencing primers designed from theoriginal sequence it is then possible to extend the sequence in bothdirections to determine the full gene sequence. Conveniently, suchsequencing is performed using denatured double stranded DNA preparedfrom a plasmid clone. Suitable techniques are described by Maniatis, T.,Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989). (see in particular Screening By Hybridization 1.90and Sequencing Denatured Double-Stranded DNA Templates 13.70).Illustrative of the invention, the polynucleotide set out in Table 1[SEQ ID NO: 1 or 3] was discovered in a DNA library derived fromStreptococcus pneumoniae 0100993.

The DNA sequence set out in Table 1 [SEQ ID NO: 1 or 3] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 [SEQ ID NO:2 or 4] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known in the art. The polynucleotide of SEQID NO: 1, between nucleotide number 288 and the stop codon which beginsat nucleotide number 1665 of SEQ ID NO: 1, encodes the polypeptide ofSEQ ID NO:2.

GlmU of the invention is structurally related to other proteins of theGlmU family, as shown by the results of sequencing the DNA encoding GlmUof the deposited strain. See PIR database S66050; Genembl D26185; andSwissprot P14192. Also see NILSSON D., HOVE-JENSEN B., ARNVIG K. MOL.GEN. GENET. 218:565-571 (1989); OGASAWARA N., NAKAI S., YOSHIKAWA H. DNARES. 1: 1-14 (1994).

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence in Table 1 [SEQ ID NO: 1 or 3]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5' and 3' sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 288 to the nucleotide immediately upstream of orincluding nucleotide 1665 set forth in SEQ ID NO: 1 of Table 1, both ofwhich encode the GlmU polypeptide.

The invention also includes polynucleotides of the formula:

    X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y

wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' endof the molecule, Y is hydrogen or a metal, R₁ and R₃ is any nucleic acidresidue, m is an integer between 1 and 3000 or zero, n is an integerbetween 1 and 3000 or zero, and R₂ is a nucleic acid sequence of theinvention, particularly a nucleic acid sequence selected from Table 1.In the polynucleotide formula above R₂ is oriented so that its 5' endresidue is at the left, bound to R₁, and its 3' end residue is at theright, bound to R₃. Any stretch of nucleic acid residues denoted byeither R group, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer. In apreferred embodiment m and/or n is an integer between 1 and 1000.

It is most preferred that the polynucleotides of the inventions arederived from Streptococcus pneumoniae, however, they may preferably beobtained from organisms of the same taxonomic genus. They may also beobtained, for example, from organisims of the same taxonomic family ororder.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae GlmUhaving an amino acid sequence set out in Table 1 [SEQ ID NO:2 or 4]. Theterm also encompasses polynucleotides that include a single continuousregion or discontinuous regions encoding the polypeptide (for example,interrupted by integrated phage or an insertion sequence or editing)together with additional regions, that also may contain coding and/ornon-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having adeduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4]. Variants thatare fragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingGlmU variants, that have the amino acid sequence of GlmU polypeptide ofTable 1 [SEQ ID NO:2 or 4] in which several, a few, 5 to 10, 1 to 5, 1to 3, 2, 1 or no amino acid residues are substituted, deleted or added,in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesand activities of GlmU.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding GlmU polypeptide having an amino acid sequence set out in Table1 [SEQ ID NO:2 or 4], and polynucleotides that are complementary to suchpolynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding GlmU polypeptide ofthe deposited strain and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthto the same are particularly preferred, and among these particularlypreferred polynucleotides, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferredamong those with at least 95%, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by a DNA of Table 1 [SEQ ID NO: 1 or 3].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.×SSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO: 1 under stringent hybridization conditions with a probehaving the sequence of said polynucleotide sequence set forth in SEQ IDNO: 1 or a fragment thereof; and isolating said DNA sequence. Fragmentsuseful for obtaining such a polynucleotide include, for example, probesand primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding GlmU and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the GlmU gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the GlmU gene may be isolated byscreening using a DNA sequence provided in Table 1 [SEQ ID NO: 1 or 3]to synthesize an oligonucleotide probe. A labeled oligonucleotide havinga sequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of Table 1 [SEQ ID NOS: 1 or 2 or 3 or 4] may be used inthe processes herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In addition to the standard A, G, C, T/U representations for nucleicacid bases, the term "N" may also be used in describing certainpolynucleotides of the invention. "N" means that any of the four DNA orRNA bases may appear at such a designated position in the DNA or RNAsequence, except it is preferred that N is not a base that when taken incombination with adjacent nucleotide positions, when read in the correctreading frame, would have the effect of generating a prematuretermination codon in such reading frame.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli , streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the GlmU polynucleotides ofthe invention for use as diagnostic reagents. Detection of GlmU in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also"individual(s)"), particularly mammals, and especially humans,particularly those infected or suspected to be infected with an organismcomprising the GlmU gene may be detected at the nucleic acid level by avariety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA, cDNA and genomic DNA may also be used in thesame ways. Using amplification, characterization of the species andstrain of prokaryote present in an individual, may be made by ananalysis of the genotype of the prokaryote gene. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the genotype of a reference sequence. Point mutationscan be identified by hybridizing amplified DNA to labeled GlmUpolynucleotide sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase digestion or bydifferences in melting temperatures. DNA sequence differences may alsobe detected by alterations in the electrophoretic mobility of the DNAfragments in gels, with or without denaturing agents, or by direct DNAsequencing. See, e.g., Myers et al., Science, 230: 1242 (1985). Sequencechanges at specific locations also may be revealed by nucleaseprotection assays, such as RNase and SI protection or a chemicalcleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA,85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA, cDNA or genomic DNA may also be used for the samepurpose, PCR or RT-PCR. As an example, PCR primers complementary to anucleic acid encoding GlmU can be used to identify and analyzemutations. Examples of representative primers are shown below in Table2.

                  TABLE 2                                                         ______________________________________                                        Primers for amplification of GlmU polynucleotides                               SEQ ID NO     PRIMER SEQUENCE                                               ______________________________________                                        5           5'-TGTCAAATTTTGCCATTATTTTAG-3'                                      6 5'-CTGGTTCTTAGGATGATGAGGAAG-3'                                            ______________________________________                                    

The invention also includes primers of the formula:

    X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y

wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' endof the molecule, Y is hydrogen or a metal, R₁ and R₃ is any nucleic acidresidue, m is an integer between 1 and 20 or zero , n is an integerbetween 1 and 20 or zero, and R₂ is a primer sequence of the invention,particularly a primer sequence selected from Table 2. In thepolynucleotide formula above R₂ is oriented so that its 5' end residueis at the left, bound to R₁, and its 3' end residue is at the right,bound to R₃. Any stretch of nucleic acid residues denoted by either Rgroup, where m and/or n is greater than 1, may be either a heteropolymeror a homopolymer, preferably a heteropolymer being complementary to aregion of a polynucleotide of Table 1. In a preferred embodiment mand/or n is an integer between 1 and 10.

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying GlmU DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving a sequence of Table 1 [SEQ ID NO: 1 or 3]. Increased or decreasedexpression of GlmU polynucleotide can be measured using any on of themethods well known in the art for the quantitation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting overexpression of GlmU protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a GlmUprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-GlmU or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by shuffling (Clackson, T. et al., (1991) Nature 352, 624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against GlmU- polypeptide may be employedto treat infections, particularly bacterial infections.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS USA 1984:81,5849).

Antagonists and Agonists--Assays and Molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of GlmUpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bactericidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising GlmU polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a GlmU agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the GlmU polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of GlmU polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in GlmU polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for GlmU antagonists is a competitive assaythat combines GlmU and a potential antagonist with GlmU-bindingmolecules, recombinant GlmU binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. GlmU can be labeled, such as byradioactivity or a colorimetric compound, such that the number of GlmUmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing GlmU-induced activities, thereby preventing the action of GlmUby excluding GlmU from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, JNeurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of GlmU.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock GlmU protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial GlmU proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

This invention provides a method of screening drugs to identify thosewhich are antibacterial by measuring the ability of the drug tointerfere with the biosynthesis of uridyl diphosphate N-acetylglucosamine by the GlmU protein.

It has been shown that E. coli GlmU protein will act as apyrophosphorylase, catalyzing the reverse reaction toN-acetylglucosamine-1-phosphate from the products of the forwardreaction, UDP-N-acetylglucosamine and pyrophosphate (Strominger, J. R.and Smith, M. S. [1959] J. Biol. Chem. 234: 1822-7). By introducing aninorganic pyrophosphatase into the reaction it will proceed in theforward direction without limit (Mengin-Lecreulx, D. and van Heijenoort,J., J. Bacteriol. 176: 5788-5795 [1994]).

In a preferred embodiment, N-acetylglucosamine-1-phosphate is incubatedwith UTP and inorganic pyrophosphatase in the presence of theS.pneumoniae GlmU protein to generate inorganic phosphate which can bemeasured calorimetrically using a suitably sensitive procedure such asthe Malachite Green method (Itaya, K. & Ui, M. Clin.Chim.Acta 14,361-366[1966) to provide a measurement of GlmU enzymatic activity. The decreaseof enzymatic activity in this reaction would indicate the presence of aninhibitor.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat diseases.

Helicobacter pylori (herein H. pylori) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France;http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofGlmU) found using screens provided by the invention, particularlybroad-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also cure gastric ulcers and gastritis.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with GlmU, or a fragment or variantthereof, adequate to produce antibody and/ or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Streptococcus pneumoniae infection. Also providedare methods whereby such immunological response slows bacterialreplication. Yet another aspect of the invention relates to a method ofinducing immunological response in an individual which comprisesdelivering to such individual a nucleic acid vector to direct expressionof GlmU, or a fragment or a variant thereof, for expressing GlmU, or afragment or a variant thereof in vivo in order to induce animmunological response, such as, to produce antibody and/ or T cellimmune response, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether thatdisease is already established within the individual or not. One way ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a GlmU or protein coded therefrom,wherein the composition comprises a recombinant GlmU or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid GlmU or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+T cells.

A GlmU polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStreptococcus pneumoniae infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the bodily fluid, preferably the blood, of theindividual; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation, may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain GlmUprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, Kits and Administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Disease(s)" means and disease caused by or related to infection by abacteria, including otitis media, conjunctivitis, pneumonia, bacteremia,meningitis, sinusitis, pleural empyema and endocarditis, and mostparticularly meningitis, such as for example infection of cerebrospinalfluid.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W. , ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M. , and Griffin, H.G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% "identity" to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5' or 3' terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously , by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO: 1or 3] was obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two ormore clones containing overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO: 1. Librariesmay be prepared by routine methods by isolating total cellular DNA fromStreptococcus pneumoniae 0100993 according to standard procedures andsize-fractionated by, for examplem, either of two methods, as follows.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, Pall, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E.coli infected with the packagedlibrary. The library is amplified by standard procedures.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 6                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2016 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  AAAAGCCTGT GCTTCAANTC TTGTGCTATA TTGGATTTTT GTTTTAAACG - #ATTGGCTG    TC    60                                                                        - -  ATTAAGTGGG CGATTAATGA TTAAAATGNA CATCATAATC CCAAAAAAAC - #TAAATAAAA    T   120                                                                         - -  AAGTGGATGA ATTTGTTTTC TCATATCTTA TAATTCTACC CTAAAAATCA - #AAAAAAATC    A   180                                                                         - -  AAAAAATGGG TTAAGGAAGA GACTTTAGAG CATTTTTTCA TTCAAGAGTG - #CGGAATGAT    T   240                                                                         - -  TGAAATATGG TATAATAAAA GGGAATTTCT ACAGAAAAGA GAAGATTATG - #TCAAATTTT    G   300                                                                         - -  CCATTATTTT AGCAGCGGGT AAAGGGACTC GCATGAAATC TGATTTGCCA - #AAAGTTTTG    C   360                                                                         - -  ACAAGGTTGC GGGTATTTCT ATGTTGGAAC ATGTTTTCCG TAGTGTGGGA - #GCTATCCAA    C   420                                                                         - -  CTGAAAAGAC AGTAACAGTT GTAGGACACA AGGCAGAATT GGTTGAGGAG - #GTCTTGGCT    G   480                                                                         - -  GACAGACAGA ATTTGTGACT CAATCTGAAC AGTTGGGAAC TGGTCATGCA - #GTTATGATG    A   540                                                                         - -  CAGAGCCTAT CTTAGAAGGT TTGTCAGGAC ACACCTTGGT CATTGCAGGA - #GATACTCCT    T   600                                                                         - -  TAATCACTGG TGAAAGCTTG AAAAACTTGA TTGATTTCCA TATCAATCAT - #AAAAATGTG    G   660                                                                         - -  CCACTATCTT GACTGCTGAA ACGGATAATC CTTTTGGCTA TGGACGAATT - #GTTCGTAAT    G   720                                                                         - -  ACAATGCTGA GGTTCTTCGT ATGGTTGAGC AGAAGGATGC TACAGATTTT - #GAAAAGCAA    A   780                                                                         - -  TCAAGGAAAT CAACACTGGA ACATACGTCT TTGACAACGA GCGTTTGTTT - #GAGGCTTTG    A   840                                                                         - -  AAAATATCAA TACCAATAAC GCTCAAGGCG AATACTATAT TACAGACGTC - #ATTGGTATT    T   900                                                                         - -  TCCGTGAAAC TGGTGAAAAA GTTGGCGCTT ATACTTTGAA AGATTTTGAT - #GAAAGTCTT    G   960                                                                         - -  GGGTAAATGA CCGTGTGGCG CTTGCGACAG CTGAGTCAGT TATGCGTCGT - #CGCATCAAT    C  1020                                                                         - -  ATAAACACAT GGTCAACGGT GTTAGCTTTG TCAATCCAAA AGCAACTTAT - #ATCGATATT    G  1080                                                                         - -  ATGTTGAGAT TGCTTCGGAA GTTCAAATCG AAGCCAATGT TACCTTGAAA - #GGGCAAACG    A  1140                                                                         - -  AAATTGGTGC TGAGACTGTT TTGACAAACG GTACTTATGT AGTGGACAGC - #ACTATCGGA    G  1200                                                                         - -  CAGGAGCGGT CATTACCAAT TCTATGATTG AGGAAAGTAG TGTTGCAGAC - #GGTGTGACA    G  1260                                                                         - -  TCGGTCCTTA TGCTCACATT CGTCCAAATT CAAGTCTGGG TGCCCAAGTT - #CATATTGGT    A  1320                                                                         - -  ACTTTGTTGA GGTGAAAGGA TCTTCAATCG GTGAGAATAC CAAGGCTGGT - #CATTTGACT    T  1380                                                                         - -  ATATCGGAAG CTGTGAAGTG GGAAGCAACG TTAATTTCGG TGCTGGAACT - #ATTACAGTC    A  1440                                                                         - -  ACTATGACGG CAAAAACAAA TACAAGACAG TCATTGGAGA CAATGTCTTT - #GTTGGTTCA    A  1500                                                                         - -  ATTCAACCAT TATTGCACCA GTAGAACTTG GTGACAATTC CCTCGTTGGT - #GCTGGTTCA    A  1560                                                                         - -  CTATTACTAA AGACGTGCCA GCAGATGCTA TTGCTATTGG TCGCGGTCGT - #CAGATCAAT    A  1620                                                                         - -  AAGACGAATA TGCAACACGT CTTCCTCATC ATCCTAAGAA CCAGTAGGAG - #CCTATCATG    G  1680                                                                         - -  AGTTTGAAGA AAAAACGCTT AGCCGAAAAG AAATCTATCA AGGACCAATA - #TTTAAACTG    G  1740                                                                         - -  TCCAAGATCA GGTTGAATTA CCAGAAGGCA AGGGAACTGC CCAACGGGAT - #TTGATTTTC    C  1800                                                                         - -  ACAATGGGGC TGTCTGTGTT TTAGCAGTAA CGGATGAACA AAAACTTATC - #TTGGTCAAG    C  1860                                                                         - -  AGTACCGCAA AGCTATCGAG GCTGTCTTTT ACGAAATTCC AGCCGGAAAA - #TTGGAAGTA    G  1920                                                                         - -  GAGAAAACAC AGCCCCTGTG GCAGCTGCCC TTCGTGAATT AGAGGAAGAA - #ACAGCCTAT    A  1980                                                                         - -  CAGGGAAATT AGAACTCTTG TACGATTTTT ATTCAG     - #                  -     #     2016                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 459 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  Met Ser Asn Phe Ala Ile Ile Leu Ala Ala - #Gly Lys Gly Thr Arg Met        1               5 - #                 10 - #                 15              - -  Lys Ser Asp Leu Pro Lys Val Leu His Lys - #Val Ala Gly Ile Ser Met                   20     - #             25     - #             30                  - -  Leu Glu His Val Phe Arg Ser Val Gly Ala - #Ile Gln Pro Glu Lys Thr               35         - #         40         - #         45                      - -  Val Thr Val Val Gly His Lys Ala Glu Leu - #Val Glu Glu Val Leu Ala           50             - #     55             - #     60                          - -  Gly Gln Thr Glu Phe Val Thr Gln Ser Glu - #Gln Leu Gly Thr Gly His       65                 - # 70                 - # 75                 - # 80       - -  Ala Val Met Met Thr Glu Pro Ile Leu Glu - #Gly Leu Ser Gly His Thr                       85 - #                 90 - #                 95              - -  Leu Val Ile Ala Gly Asp Thr Pro Leu Ile - #Thr Gly Glu Ser Leu Lys                   100     - #            105     - #            110                 - -  Asn Leu Ile Asp Phe His Ile Asn His Lys - #Asn Val Ala Thr Ile Leu               115         - #        120         - #        125                     - -  Thr Ala Glu Thr Asp Asn Pro Phe Gly Tyr - #Gly Arg Ile Val Arg Asn           130             - #    135             - #    140                         - -  Asp Asn Ala Glu Val Leu Arg Met Val Glu - #Gln Lys Asp Ala Thr Asp       145                 - #150                 - #155                 -         #160                                                                             - -  Phe Glu Lys Gln Ile Lys Glu Ile Asn Thr - #Gly Thr Tyr Val Phe        Asp                                                                                              165 - #                170 - #                175            - -  Asn Glu Arg Leu Phe Glu Ala Leu Lys Asn - #Ile Asn Thr Asn Asn Ala                   180     - #            185     - #            190                 - -  Gln Gly Glu Tyr Tyr Ile Thr Asp Val Ile - #Gly Ile Phe Arg Glu Thr               195         - #        200         - #        205                     - -  Gly Glu Lys Val Gly Ala Tyr Thr Leu Lys - #Asp Phe Asp Glu Ser Leu           210             - #    215             - #    220                         - -  Gly Val Asn Asp Arg Val Ala Leu Ala Thr - #Ala Glu Ser Val Met Arg       225                 - #230                 - #235                 -         #240                                                                             - -  Arg Arg Ile Asn His Lys His Met Val Asn - #Gly Val Ser Phe Val        Asn                                                                                              245 - #                250 - #                255            - -  Pro Lys Ala Thr Tyr Ile Asp Ile Asp Val - #Glu Ile Ala Ser Glu Val                   260     - #            265     - #            270                 - -  Gln Ile Glu Ala Asn Val Thr Leu Lys Gly - #Gln Thr Lys Ile Gly Ala               275         - #        280         - #        285                     - -  Glu Thr Val Leu Thr Asn Gly Thr Tyr Val - #Val Asp Ser Thr Ile Gly           290             - #    295             - #    300                         - -  Ala Gly Ala Val Ile Thr Asn Ser Met Ile - #Glu Glu Ser Ser Val Ala       305                 - #310                 - #315                 -         #320                                                                             - -  Asp Gly Val Thr Val Gly Pro Tyr Ala His - #Ile Arg Pro Asn Ser        Ser                                                                                              325 - #                330 - #                335            - -  Leu Gly Ala Gln Val His Ile Gly Asn Phe - #Val Glu Val Lys Gly Ser                   340     - #            345     - #            350                 - -  Ser Ile Gly Glu Asn Thr Lys Ala Gly His - #Leu Thr Tyr Ile Gly Ser               355         - #        360         - #        365                     - -  Cys Glu Val Gly Ser Asn Val Asn Phe Gly - #Ala Gly Thr Ile Thr Val           370             - #    375             - #    380                         - -  Asn Tyr Asp Gly Lys Asn Lys Tyr Lys Thr - #Val Ile Gly Asp Asn Val       385                 - #390                 - #395                 -         #400                                                                             - -  Phe Val Gly Ser Asn Ser Thr Ile Ile Ala - #Pro Val Glu Leu Gly        Asp                                                                                              405 - #                410 - #                415            - -  Asn Ser Leu Val Gly Ala Gly Ser Thr Ile - #Thr Lys Asp Val Pro Ala                   420     - #            425     - #            430                 - -  Asp Ala Ile Ala Ile Gly Arg Gly Arg Gln - #Ile Asn Lys Asp Glu Tyr               435         - #        440         - #        445                     - -  Ala Thr Arg Leu Pro His His Pro Lys Asn - #Gln                               450             - #    455                                                - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1300 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  TCATGCAGTT ATGATGACAG AGCCTATCTT AGAAGGTTTG TCAGGACACA - #CCTTGGTCA    T    60                                                                         - -  TGCAGGAGAT ACTCCTTTAA TCACTGGTGA AAGCTTGAAA AACTTGATTG - #ATTTCCATA    T   120                                                                         - -  CAATCATAAA AATGTGGCCA CTATCTTGAC TGCTGAAACG GATAATCCTT - #TTGGCTATG    G   180                                                                         - -  ACGAATTGTT CGTAATGACA ATGCTGAGGT TCTTCGTATG GTTGAGCAGA - #AGGATGCTA    C   240                                                                         - -  AGATTTTGAA AAGCAAATCA AGGAAATCAA CACTGGAACA TACGTCTTTG - #ACAACGAGC    G   300                                                                         - -  TTTGTTTGAG GCTTTGAAAA ATATCAATAC CAATAACGCT CAAGGCGAAT - #ACTATATTA    C   360                                                                         - -  AGACGTCATT GGTATTTTCC GTGAAACTGG TGAAAAAGTT GGCGCTTATA - #CTTTGAAAG    A   420                                                                         - -  TTTTGATGAA AGTCTTGGGG TAAATGACCG TGTGGCGCTT GCGACAGCTG - #AGTCAGTTA    T   480                                                                         - -  GCGTCGTCGC ATCAATCATA AACACATGGT CAACGGTGTT AGCTTTGTCA - #ATCCAAAAG    C   540                                                                         - -  AACTTATATC GATATTGATG TTGAGATTGC TTCGGAAGTT CAAATCGAAG - #CCAATGTTA    C   600                                                                         - -  CTTGAAAGGG CAAACGAAAA TTGGTGCTGA GACTGTTTTG ACAAACGGTA - #CTTATGTAG    T   660                                                                         - -  GGACAGCACT ATCGGAGCAG GAGCGGTCAT TACCAATTCT ATGATTGAGG - #AAAGTAGTG    T   720                                                                         - -  TGCAGACGGT GTGACAGTCG GTCCTTATGC TCACATTCGT CCAAATTCAA - #GTCTGGGTG    C   780                                                                         - -  CCAAGTTCAT ATTGGTAACT TTGTTGAGGT GAAAGGATCT TCAATCGGTG - #AGAATACCA    A   840                                                                         - -  GGCTGGTCAT TTGACTTATA TCGGAAGCTG TGAAGTGGGA AGCAACGTTA - #ATTTCGGTG    C   900                                                                         - -  TGGAACTATT ACAGTCAACT ATGACGGCAA AAACAAATAC AAGACAGTCA - #TTGGAGACA    A   960                                                                         - -  TGTCTTTGTT GGTTCAAATT CAACCATTAT TGCACCAGTA GAACTTGGTG - #ACAATTCCC    T  1020                                                                         - -  CGTTGGTGCT GGTTCAACTA TTACTAAAGA CGTGCCAGCA GATGCTATTG - #CTATTGGTC    G  1080                                                                         - -  CGGTCGTCAG ATCAATAAAG ACGAATATGC AACACGTCTT CCTCATCATC - #CTAAGAACC    A  1140                                                                         - -  GTAGGAGCCT ATCATGGAGT TTGAAGAAAA AACGCTTAGC CGAAAAGAAA - #TCTATCAAG    G  1200                                                                         - -  ACCAATATTT AAACTGGTCC AAGATCAGGT TGAATTACCA GAAGGCAAGG - #GAACTGCCC    A  1260                                                                         - -  ACGGGATTTG ATTTTCCACA ATGGGGCTGT CTGTGTTTTA    - #                      - #  1300                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 380 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  His Ala Val Met Met Thr Glu Pro Ile Leu - #Glu Gly Leu Ser Gly His        1               5 - #                 10 - #                 15              - -  Thr Leu Val Ile Ala Gly Asp Thr Pro Leu - #Ile Thr Gly Glu Ser Leu                   20     - #             25     - #             30                  - -  Lys Asn Leu Ile Asp Phe His Ile Asn His - #Lys Asn Val Ala Thr Ile               35         - #         40         - #         45                      - -  Leu Thr Ala Glu Thr Asp Asn Pro Phe Gly - #Tyr Gly Arg Ile Val Arg           50             - #     55             - #     60                          - -  Asn Asp Asn Ala Glu Val Leu Arg Met Val - #Glu Gln Lys Asp Ala Thr       65                 - # 70                 - # 75                 - # 80       - -  Asp Phe Glu Lys Gln Ile Lys Glu Ile Asn - #Thr Gly Thr Tyr Val Phe                       85 - #                 90 - #                 95              - -  Asp Asn Glu Arg Leu Phe Glu Ala Leu Lys - #Asn Ile Asn Thr Asn Asn                   100     - #            105     - #            110                 - -  Ala Gln Gly Glu Tyr Tyr Ile Thr Asp Val - #Ile Gly Ile Phe Arg Glu               115         - #        120         - #        125                     - -  Thr Gly Glu Lys Val Gly Ala Tyr Thr Leu - #Lys Asp Phe Asp Glu Ser           130             - #    135             - #    140                         - -  Leu Gly Val Asn Asp Arg Val Ala Leu Ala - #Thr Ala Glu Ser Val Met       145                 - #150                 - #155                 -         #160                                                                             - -  Arg Arg Arg Ile Asn His Lys His Met Val - #Asn Gly Val Ser Phe        Val                                                                                              165 - #                170 - #                175            - -  Asn Pro Lys Ala Thr Tyr Ile Asp Ile Asp - #Val Glu Ile Ala Ser Glu                   180     - #            185     - #            190                 - -  Val Gln Ile Glu Ala Asn Val Thr Leu Lys - #Gly Gln Thr Lys Ile Gly               195         - #        200         - #        205                     - -  Ala Glu Thr Val Leu Thr Asn Gly Thr Tyr - #Val Val Asp Ser Thr Ile           210             - #    215             - #    220                         - -  Gly Ala Gly Ala Val Ile Thr Asn Ser Met - #Ile Glu Glu Ser Ser Val       225                 - #230                 - #235                 -         #240                                                                             - -  Ala Asp Gly Val Thr Val Gly Pro Tyr Ala - #His Ile Arg Pro Asn        Ser                                                                                              245 - #                250 - #                255            - -  Ser Leu Gly Ala Gln Val His Ile Gly Asn - #Phe Val Glu Val Lys Gly                   260     - #            265     - #            270                 - -  Ser Ser Ile Gly Glu Asn Thr Lys Ala Gly - #His Leu Thr Tyr Ile Gly               275         - #        280         - #        285                     - -  Ser Cys Glu Val Gly Ser Asn Val Asn Phe - #Gly Ala Gly Thr Ile Thr           290             - #    295             - #    300                         - -  Val Asn Tyr Asp Gly Lys Asn Lys Tyr Lys - #Thr Val Ile Gly Asp Asn       305                 - #310                 - #315                 -         #320                                                                             - -  Val Phe Val Gly Ser Asn Ser Thr Ile Ile - #Ala Pro Val Glu Leu        Gly                                                                                              325 - #                330 - #                335            - -  Asp Asn Ser Leu Val Gly Ala Gly Ser Thr - #Ile Thr Lys Asp Val Pro                   340     - #            345     - #            350                 - -  Ala Asp Ala Ile Ala Ile Gly Arg Gly Arg - #Gln Ile Asn Lys Asp Glu               355         - #        360         - #        365                     - -  Tyr Ala Thr Arg Leu Pro His His Pro Lys - #Asn Gln                           370             - #    375             - #    380                         - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  ATGTCAAATT TTGCCATTAT TTTAG         - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -  CTGGTTCTTA GGATGATGAG GAAG         - #                  - #                    24                                                                    __________________________________________________________________________

What is claimed is:
 1. A recombinant polynucleotide segment comprisingnucleotides 288 to 1665 of the polynucleotide sequence set forth in SEQID NO:1, or the full complement of nucleotides 288 to 1665 of thepolynucleotide sequence set forth in SEQ ID NO:1.
 2. A recombinantpolynucleotide segment, wherein the recombinant polynucleotide segment(a) encodes a polypeptide comprising the amino acid sequence of SEQ IDNO:2, or (b) is the full complement of the entire length of apolynucleotide sequence that encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:2.
 3. A vector comprising the polynucleotidesegment of claim 2, which segment encodes the polypeptide.
 4. Anisolated host cell transfected with the polynucleotide segment of claim2 to express the polynucleotide sequence.
 5. A process for producing aGlmU polypeptide of the polynucleotide sequence comprising the step ofculturing a host cell of claim 4 under conditions sufficient for theproduction of said polypeptide.
 6. An isolated polynucleotide segmentcomprising: a first polynucleotide sequence, or the full complement ofthe entire length of the first polynucleotide sequence, wherein thefirst polynucleotide sequence hybridizes to the complement of a nucleicacid sequence that encodes the amino acid sequence set forth in SEQ IDNO:2 or 4, wherein the hybridization conditions include incubation at42° C. in a solution comprising 50% formamide, 5× SSC (150 mN NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 microgras/ml denatured, shearedsalmon sperm DNA, followed by washing in 0.1× SSC at about 65° C.
 7. Theisolated polynucleotide segment of claim 6, wherein the isolatedpolynucleotide segment comprises the first polynucleotide sequence. 8.The isolated polynucleotide segment of claim 7, wherein the isolatedpolynucleotide segment comprises the first polynucleotide sequence,wherein the first polynucleotide sequence encodes a polypeptide selectedfrom the group consisting of:(a) a polypeptide comprising SEQ ID NO 2;(b) a polypeptide comprising SEQ ID NO:4; (c) an amino acid sequencecomprising a portion of the polypeptide of (a) or (b) wherein theportion contains at least 50 amino acids; and, (d) an amino acidsequence comprising a portion of the polypeptide of (a) or (b) whereinthe portion contains at least 30 amino acids.
 9. The isolatedpolynucleotide segment of claim 6, wherein the isolated polynucleotidesegment comprises the full complement of the entire length of the firstpolynucleotide sequence.
 10. The isolated polynucleotide segment ofclaim 9, wherein the first polynucleotide sequence encodes apolypeptide, wherein the polypeptide is selected from the groupconsisting of:(a) a polypeptide comprising SEQ ID NO:2; (b) apolypeptide comprising SEQ ID NO:4; (c) an amino acid sequencecomprising a portion of the polypeptide of (a) or (b) wherein theportion contains at least 50 amino acids; and, (d) an amino acidsequence comprising a portion of the polypeptide of (a) or (b) whereinthe portion contains at least 30 amino acids.
 11. A vector comprisingthe isolated polynucleotide segment of claim
 6. 12. An isolated hostcell transfected with the isolated polynucleotide segment of claim 7 toexpress the first polynucleotide sequence.
 13. A process for producing apolypeptide of the first polynucleotide sequence comprising the step ofculturing the host cell of claim 12 under conditions sufficient for theproduction of said polypeptide, which is encoded by the firstpolynucleotide sequence.
 14. An isolated polynucleotide segmentcomprising a first polynucleotide sequence, or the full complement ofthe entire length of such first polynucleotide sequence, wherein thefirst polynucleotide sequence hybridizes to the complement of a nucleicacid sequence which encodes the same mature polypeptide, expressed bythe GlmU gene contained in Streptococcus pneumoniae 0100993 contained inNCIMB Deposit No. 40794; wherein the hybridization conditions includeincubation at 42° C. in a solution comprising 50% formamide, 5× SSC (150mN NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 micrograms/mldenatured, sheared salmon sperm DNA, followed by washing in 0.1× SSC atabout 65° C.
 15. An isolated polynucleotide segment comprising apolynucleotide sequence that encodes the same mature polypeptideexpressed by the GlmU gene contained in Streptococcus pneumoniae 0100993contained in NCIMB Deposit No.
 40794. 16. A polynucleotide which encodesa fusion polypeptide and which includes the isolated polynucleotidesegment according to claim
 14. 17. An isolated polynucleotide segmentcomprising a first polynucleotide wherein the first polynucleotidesequence hybridizes to the complement of SEQ ID NO:1, wherein thehybridization conditions include incubation at 42° C. in a solutioncomprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 micrograms/ml denatured, sheared salmon spermDNA, followed by washing in 0.1× SSC at about 65° C.
 18. A vectorcomprising the isolated polynucleotide segment of claim
 17. 19. Anisolated host cell comprising the vector of claim
 18. 20. An isolatedpolynucleotide segment comprising a first polynucleotide wherein thefirst polynucleotide sequence hybridizes to the complement of SEQ IDNO:3, wherein the hybridization conditions include incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 nM NaCl 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing in 0.1× SSC at about 65° C.
 21. Avector compress the isolated polynucleotide segment of claim
 20. 22. Anisolated host cell comprising the vector of claim
 21. 23. A compositioncomprising the isolated polynucleotide of claim 6, which polynucleotideis according to the formula;

    X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y

wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' endof the molecule, Y is hydrogen or a metal, each occurrence of R₁ and R₃is independently any nucleic acid residue, m is an integer between 1 and3000 or zero, n is an integer between 1 and 3000 or zero, and R₂ is thefirst polynucleotide sequence of claim 8.